Repeated injections with abo- or onabotulinumtoxin type A  may lead to induction of neutralizing antibodies (NABs) and/or a secondary treatment failure (STF). The relation between NABs and STF is still unclear.To demonstrate that a significant improvement can be observed in patients with STF after abo- or onaBoNT/A-treatment when switched to incobotulinumtoxin type A (incoBoNT/A) and that in NAB-positive patients without STF abo- or onaBoNT/A-treatment can be continued without significant worsening.Paralysis times (PT) of the mouse hemidiaphragm assay (MHDA) and clinical outcome (TSUI-score) was analyzed in 60 patients with cervical dystonia (CD) and STF after abo- or onaBoNT/A-treatment (STF-group) who were switched to incobotulinumtoxin type A (incoBoNT/A). These data were compared to those of 34 patients who were exclusively treated with incoBoNT/A (INCO-group). Furthermore, PTs and TSUI-scores were followed up over 7 years in 9 patients with NABs but without STF who were switched to inco-BoNT/A (SWI-group) and 9 other patients with NABs who remained on their previous BoNT/A preparation (NO-SWI-group).In the STF-group, a significant improvement of TSUI-scores could be detected after switch to incoBoNT/A. This improvement was less pronounced than in the INCO-group. There was no significant difference in long-term outcome between the SWI- and NO-SWI-group.The best strategy is to avoid the induction of NABs. A switch to incoBoNT/A may lead to improvement in patients with STF. However, in some patients with NABs without STF, BoNT/A-treatment can be continued without significant worsening. Injections with botulinum neurotoxin type A (BoNT/A) are an effective and safe treatment for a variety of neurological and non-neurological indications , 2. For It seems to be common sense that NAB formation occurs only in a small percentage of BoNT/A-treated patients , 4. UnfoSo far, only a few cross-sectional studies on NAB formation in BoNT/A long-term treated patients are available. In still-responding patients with cervical dystonia (CD) , and in Prevalence divided by mean duration of treatment allows a rough estimation of the incidence of NAB formation. The incidences reported so far for ona- and abobotulinumtoxin type A  vary between 0.5 and 2.5%/year –11. TakiNevertheless, the relationship between secondary non-responsiveness or secondary treatment failure (STF) and NABs is unclear , 4. The On the other hand, in clinical practice, in many centers, the outcome is not or barely routinely controlled by means of simple assessment scales  which are insensitive to subtle changes of outcome. This is similar to the result of the mouse lethal assay (MLA) which is either positive or negative. Such discrete parameters are not suitable for correlation analysis between clinical findings and NAB-testing.But even when well-established scales as the TSUI-score  or the CTo contribute further to the analysis of this complex relationship between clinical outcome and antibody titres, the present study was performed correlating the results of MHDA-testing and treatment-related data of a cross-sectional study on 60 clinically well-characterized CD-patients with STF who had been switched to incobotulinumtoxinA (incoBoNT/A). Furthermore, a small, longitudinal study is added comparing MHDA-test results and clinical data in 18 still-responding CD-patients who had been tested in 2010, 2013, and 2017. Nine of these 18 patients had been switched to incoBoNT/A, 9 remained on their previous BoNT/A preparation even after the detection of NABs.The present study was performed according to the guidelines for good clinical practice (GCP) and according to the declaration of Helsinki. It was approved by the local ethics committee of the University of Düsseldorf (number: 4085). The presented data are parts of the medical theses of Beyza Ürer (BÜ) and Raphaela Brauns (RB).In 2017, BÜ screened more than 120 charts of patients with CD, who were regularly treated in the outpatient department of the University hospital at Düsseldorf (Germany) and in whom their initial BoNT/A preparation had been switched to another BoNT/A preparation during their course of treatment in Düsseldorf because of the development of a partial or complete secondary treatment failure (switchers). Switchers who fulfilled the following inclusion and exclusion criteria were informed on the purpose of the study while they were waiting for the next BoNT/A in the botulinum toxin ambulance.Inclusion criteria of the study were: (i) age over 17, (ii) diagnosis of idiopathic CD, (iii) continuous treatment in the botulinum toxin ambulance every 12–13 weeks without interruption of BoNT therapy of more than one treatment cycle, and (iv) at least 3 injections of BoNT. Exclusion criteria were: (i) patient under legal care, (ii) multifocal, segmental, and/or symptomatic dystonia at the onset of BoNT therapy, and (iii) additional disabling disease other than CD. Especially, patients with clinical manifest disturbances of mood and perception were excluded.n = 48) and the ONA-group (n = 11). In one patient, the initial BoNT/A preparation had not been documented.Sixty patients gave informed written consent and were consecutively recruited (STF-group). Depending on the initial BoNT/A preparation (aboBoNT/A or onaBoNT/A), switchers were split up into the ABO-group .During the screening of the charts, BÜ detected nine still-responding CD-patients who had participated in a cross-sectional study in 2010  and had ®-table with 31 rows (corresponding to the days of the month) and 21 columns ranging from 0 up to 100% in 5% steps had to be completed.Patients in our BoNT ambulance are trained to assess the remaining severity of CD in percent of the severity of CD at onset of BoNT therapy every day. For each month, either a prepared table on a sheet of paper or an ExelOn the day of recruitment, patients were asked for the change of the severity of CD in percent of the severity of CD at the onset of BoNT/A therapy (IMPQ). Furthermore, they had to mark the actual severity of CD (ASCD) on a line of about 15 cm length with 0 indicating complete relief of symptoms and 10 cm indicating the severity of CD at the onset of BoNT/A therapy. A value shorter than 10 indicated improvement, and a value larger than 10 indicated worsening. The value 10*(10-ASCD) was the percentage of the change of the severity of CD calculated from this visual analogue scale (IMPD). The treating physician scored the actual severity of CD at the day of recruitment by means of the TSUI-score  and documented the BoNT/A preparation used as well as the actual total dose (ADOSE).The following demographical data were extracted from the charts: age at the day of recruitment (AGE), age at onset of symptoms (AOS), age at onset of therapy (AOT), and duration of therapy (DURT). The time span during which patients had tolerated symptoms without BoNT therapy was determined (DURS = AOT-AOS). BÜ and RB also extracted the best TSUI-score (BTSUI) documented in the charts of the recruited patients and the time span from the onset of symptoms to BTSUI (TTB).TSUI-score at onset of therapy (ITSUI), initial BoNT-preparation, and initial total dose (IDOSE) were extracted from the charts. Improvement of the severity of CD on the basis of the TSUI-score (IMPTSUI) was calculated as (ITSUI-ATSUI) *100/ITSUI). For sake of comparison, doses of different preparations were transformed into unified dose units (uDU) by multiplying ona- and incoBoNT/A doses by 3 and leaving aboBoNT/A doses unchanged following evidence-based data and a European consensus paper . The incAlternative therapies as acupuncture, physiotherapy, etc. were not controlled in the present study.n = 112 = 60 + 34 + 18) were coded and sent to a blinded contractor  for MHDA-testing. All samples were analyzed in a batch and a complete list of MHDA-test results  was returned.On the day of recruitment, blood samples were taken and deeply frozen. After recruitment of all patients, all blood samples .Patients were split up into an ABO-, ONA-, and INCO-subgroup. A Chi-square analysis was performed to whether sex distribution and NAB frequency were different across patient subgroups. A three-group repeated measurement (rm)-ANOVA was performed to test whether AGE, AOS, DURS, DURT, IDOSE, ADOSE, INDOSE, ITSUI, BTSUI, ATSUI, IMPTSUI, IMPQ, and IMPD were different between the subgroups. Pearson and Spearman’s rho correlation coefficients were determined for the paralysis time and clinical data . Register operational characteristics (ROC)-curves of IMPQ, IMPD, IMPTSUI, ATSUI, and ADOSE analyzing sensitivity and specificity for MHDA-positivity were calculated. All statistical analyses were performed using the SPSSPatients were trained to assess the effect of treatment by scoring daily the severity of CD in percent of the initial severity of CD at the onset of BoNT therapy ). Three examples of PGA-curves of the first 4 incoBoNT/A injections are presented. The black dots in Fig. ® (SD 113); ABO-group: mean 620 U Dysport® (SD 150); INCO-group: mean 189 U Xeomin® (SD 73) did not differ significantly. With ongoing BoNT therapy, the TSUI-score significantly (p < 0.001) improved in all three patient subgroups.The initial severity of CD (ITSUI) at the onset of BoNT therapy was about the same in the ABO- and ONA-group . In the small ONA-group (full squares), mean BTSUI was 3.7 and reached after 36 months (S.D. 24.6 months). Doses had been increased, but did not differ at TTB across the three patient groups (ONA-group 321 U Botox® (S.D. 229); ABO-group: 843 U Dysport® (S.D. 697); INCO-group: 267 U Xeomin® (S.D. 90). In all three subgroups, TTB was highly variable and the shortest in the INCO- and the longest in the ABO-group. Because of the high variability, TTB did not differ significantly between the three patient subgroups.Mean best TSUI-score (BTSUI) was the lowest (1.7/S.D. 1.7) in the INCO-group ) and that in the ABO-group (mean: 265 U Xeomin® (S.D. 73) were nearly identical to BDOSE in the INCO-group.After an initial good response, the switchers in the ABO- and ONA-group developed a partial or complete secondary treatment failure and severity of CD worsened again until patients were switched to incoBoNT/A. After 7.9 years of aboBoNT/A-treatment in the mean (S.D. 5.6 years), mean TSUI increased up to 7.8 (S.D. 3.1). After 7.3 years of onaBoNT/A-treatment in the mean (S.D. 4.3 years), mean TSUI increased up to 8.4 (S.D. 3.6). On the day of switch to incoBoNT/A, TSUI-score (STSUI) did not significantly differ from ITSUI in the ABO- and in the ONA-group. Initial incoBoNT/A dose in the ONA- (mean: 245 U Xeomin® (S.D. 92) in the ABO-group. The TSUI-score decreased significantly (p < 0.005) to 5.32 . In the ONA-group, incoBoNT/A dose had been increased to 370 U Xeomin® (S.D. 87) after 8.8 years (S.D. 2.7 years). The TSUI-score significantly (p < 0.05) decreased to 6.82 .After 7.2 years (S.D. 3.1 years) of incoBoNT/A-treatment, incoBoNT/A dose had been increased to 349 U Xeomin® (S.D. 91). ADOSE did not differ between the three patient groups. Mean ATSUI was 3.3 (S.D. 2.4) in the INCO-group which is even lower than mean BTSUI and by far lower than mean ATSUI in the ABO- and ONA-group.After 6.3 years of incoBoNT/A therapy (S.D. 2.3), incoBoNT/A dose was increased to 305 U Xeomin2-testing yielded a significant difference between the prevalence of MHDA-positive patients in the ABO- compared to the INCO-group, but not between ONA- and INCO-group or ABO- and ONA-group.On the day of recruitment, only 1 patient (9.1%) was MHDA-positive in the ONA-group and 14 patients (= 26.1%) were MHDA-positive in the ABO-group. In the INCO-group, no patient had a positive MHDA-test. ChiWhen relative frequencies of MHDA-positive patients in the entire STF-group (ABO- plus ONA-group) were calculated for TSUI-ranges with a bin-width of 2 TSUI-points a non-linear increase of the prevalence of MHDA-positive patients was found up to a TSUI-range of 11–12 Fig. . Above tr = 0.135, p = 0.393, n.s.). In the MHDA-positive switchers . When the regression line was calculated for all patients, a clear tendency, but not a significant correlation was found . However, Spearman’s rho rank correlation coefficient between PT and TSUI-score was significant  for the entire STF-group. Good or even better non-parametric correlations were found between PT and IMPQ or IMPD .When PT and TSUI-scores were correlated in the MHDA-negative switchers  or after  and in the 9 switchers  yielded regression lines which looked very similar and had a positive slope. The regression line between TSUI-score and PT in all 18 patients . This corresponds to the missing correlation between TSUI-scores and PT in the entire STF-group (see results part 4).Correlation between TSUI-scores ) yielded a negative slope, whereas in the 9 switchers  a positive slope. The regression line between TSUI-score and PT in all 18 patients .Correlation between TSUI-scores and PT of the year 2017 .Correlation of the changes of PT (PT2017-PT2010) with TSUI-scores of the year 2010 yielded a significantly negative correlation  which was even more pronounced in the switchers (6.44*(TSUI2017-TSUI2010) + 10.66; r = 0.628; p < 0.05, 1-sided testing), whereas the corresponding regression line in the non-switchers had a negative slope (− 2.24(TSUI2017-TSUI2010) + 1.25; r = − 0.151, n.s.).Correlation of the differences of the TSUI-score between 2017 and 2010 and the differences of the paralysis times  does not provide appropriate information on antibody titers and does not allow the calculation of a parametric correlation between clinical outcome and antibody titers. Furthermore, the technical details of the MHDA are difficult to maintain constant over years. Therefore, it is difficult to compare antibody titers over years.These are further reasons why it is difficult to demonstrate a significant relationship between clinical outcomes and NAB titers.It is difficult to detect a significant correlation between the paralysis time as an outcome measure of the MHDA and clinical outcome measures. However, the more refined the clinical outcome measure is, the more significant is this relationship. Patient’s assessment of the improvement was documented on a scale with 21 steps between 0 and 100%, and physicians rated the severity of CD by means of the TSUI-score which usually varies between 4 and 16 with a worse resolution to detect subtle changes of disease severity. This explains why the sensitivity to predict a positive MHDA-test result of IMPQ or IMPD was higher than that of ATSUI Fig.  and IMPTThe MHDA-test reliably detects a decrease of NAB titers when NABs have been induced under abo- or onaBoNT/A and BoNT/A therapy is switched to incoBoNT/A. This has been demonstrated in a previous study , 17, is This is poorly understood. Whether cellular immunological reactions to BoNT therapy may play a role in addition to antibody-mediated immunological processes has not been excluded so far. And whether a botulinum neurotoxin molecule can still be taken up into a presynaptic nerve terminal of a mouse muscle despite a human antibody being bound to a special epitope of the BoNT molecule has also not been excluded. This would imply that human NAB titers were determined or estimated too low in the MHDA.As demonstrated in Fig. The present study describes the largest cohort of patients with secondary treatment failure in whom BoNT/A preparation is switched from a complex containing to a complex free BoNT/A preparation. Furthermore, it presents data on the follow-up of NAB titers in still-responding patients in whom BoNT/A preparation has not been switched. A highly significant correlation between NAB titers and clinical outcomes was detected. NABs and clinical outcome possibly follow a complex hysteresis regulation process. However, this is highly speculative. We, therefore, recommend a well-designed multi-center long-term study in future to analyze the temporal development of clinical outcome and NAB induction and to provide a better data basis for the understanding of the difficulty to demonstrate a significant relationship between NAB titers and clinical outcome.


This paper aimed to analyze IncRNA SOX2-OT expression in patients with carotid atherosclerosis and to elucidate the predictive significance of SOX2-OT on carotid atherosclerosis.The levels of SOX2-OT from 185 participants were tested. The relationship between CIMT levels and SOX2-OT expression was examined by Pearson analysis. The clinical value of SOX2-OT was investigated by the ROC curve, K–M curve, and COX regression analysis. The comparison of SOX2-OT expression between patients with good prognosis and poor prognosis was also performed.The expression of SOX2-OT was augmented in the patients with carotid atherosclerosis and was correlated with the level of CIMT. The high level of SOX2-OT might be a risk factor for carotid atherosclerosis. An enhancement of SOX2-OT expression was found in patients with poor prognosis. SOX2-OT might be an independent prognostic biomarker.SOX2-OT was upregulated in patients with carotid atherosclerosis and might be a predictive indicator in the progression of carotid atherosclerosis. Atherosclerosis is a disorder formed by fibrofatty lesions in the artery wall . CarotidThe regulatory effect of non-coding RNAs on atherosclerosis has been confirmed by many studies. Long non-coding RNA (lncRNA) MATERIAL, lncRNA MIAT, and lncRNA H19 can affect the processes of lipoprotein uptake, cholesterol reverse transport, cell inflammation, and angiogenesis –10. MicrOur hypothesis was that SOX2-OT might play roles in atherosclerosis and possess clinical values for patients with atherosclerosis. Thus, we aimed to assess the clinical significance of SOX2-OT in atherosclerosis. For this purpose, the expression of SOX2-OT was evaluated in patients with atherosclerosis. The association between CIMT and SOX2-OT was provided by the Pearson correlation. The diagnostic role of SOX2-OT was tested by the ROC curve. The prognosis significance was unvealed by the K–M curve and Cox regression.According to the inclusion criteria and exclusion criteria, 95 patients with asymptomatic carotid atherosclerosis from Shanghai Sixth People’s Hospital Affiliated to Shanghai JiaoTong University were selected for carotid ultrasound examination to evaluate the degree of carotid atherosclerosis. The ultrasonic diagnostic instrument model was ATL HDI 3000 ultrasound system (Advanced Technology Laboratories). All eligible patients meet the following inclusion criteria: carotid intima-media thickness (CIMT) of 0.9–1.2 mm . The excAll participants were followed up for 5 years. The occurrence of adverse cardiovascular events, such as death, unstable angina pectoris, heart failure, and nonfatal myocardial infarction were recorded. The endpoint events were diagnostic according to the cardiovascular endpoint events guideline of ACC/AHA .The total RNA was isolated from serum sample using the EZ-press serum/plasma RNA purification kit (EZBioscience). That is, the serum sample and the lysis solution were fully mixed and lysed, and the supernatant was collected. Then, the reagent and ethanol were mixed and transfer to an RNA binding column. The eluent reagent was used to remove impurities efficiently and purify total RNA by subsequent washing.−ΔΔCt formula.The gDNA remover (EZBioscience) was utilized to remove the genomic DNA residues in the RNA extraction process, and the treated RNA samples were directly used for subsequent experiments. The reverse transcription system  was used to synthesize first-strand cDNA from the total RNA. Then, the realstar green fast mixture  was used for assessed relative expression of SOX2-OT. That is, 50 ng cDNA was mixed with 1 µl primers (10 µM), 25 µl 2×RealStar Fast SYBR qPCR Mix, 1 µl ROX Reference Dye, and Sterile Water. After that, this mixture was put on the ABI 7500 system. The expression of serum GAPDH was detected as an internal reference. The expression was processed using the 22 test was used for counting data. The clinical values of SOX2-OT were predicted by the Pearson correlation, ROC curve, K–M curve following log-rank test, and COX regression test. COX regression analysis was used to analyze the influencing factors of atherosclerosis. Among them, P < 0.05 was thought that the difference was statistically significant.According to 5% of false positive error rate , power = 90%, beta = 0.1, and dropout rate = 20%, PASS 11.0 was utilized to estimate the sample size, and at least 70 subjects in each group should be the sample size limit. All the data were analyzed by SPSS 26.0 statistical software. T-test was used to compare the measurement data between groups, χP < 0.05). There were no significant differences on age, sex, drinking, smoking, diabetes, and HDL-C between the two groups (Table P > 0.05).The age of 95 patients with carotid atherosclerosis was 60.01 ± 7.83 years old (Table P < 0.001).All serum specimens of participants were applied in the RT-qPCR to analyze the expression of SOX2-OT. The serum SOX2-OT level in the atherosclerosis group was significantly higher than that in the control group .Based on the abnormality of SOX2-OT expression in patients with atherosclerosis, Pearson association analysis was performed to elucidate the interconnection between SOX2-OT expression and CIMT levels. As shown in Fig. The possible utility of serum SOX2-OT as a biomarker for atherosclerosis patients was evaluated. As exhibited in Fig. P < 0.001).All patients were divided into poor outcomes and good outcomes according to the situation of a 5-year follow-up. There were 19 patients with endpoint events, and 76 patients without. The expression of SOX2-OT was declined in the good prognosis patients compared with the poor prognosis patients .The prognostic importance of serum SOX2-OT in patients with atherosclerosis was tested by K–M analysis. A total of 42 patients was divided into low expression group and 53 patients were recruited in the high expression group in accordance with the median expression of SOX2-OT. The finding showed that the occurrence of endpoint events in the low expression of SOX2-OT was lower than that in the high expression of the SOX2-OT group , CIMT , and SOX2-OT  were independent risk factors for patients with atherosclerosis (Table In COX regression analysis, the incidence of cardiovascular events in five years was taken as dependent variables, and age, sex, drinking, smoking, hypertension, diabetes, HDL-C, LDL-C, CRP, CIMT, and serum SOX2-OT were taken as independent variables. The results showed that CRP (95% CI = 1.129–15.157, HR = 4.137, Asymptomatic carotid atherosclerosis, which has no history of cardio-cerebral vascular events, is an early stage of atherosclerotic disease . CarotidLncRNAs are widely distributed in animals, plants, viruses, and other organisms, and they can play roles in gene expression regulation, cell proliferation, apoptosis, and metabolism. Many studies have confirmed that IncRNAs can affect the occurrence and development of atherosclerosis at the epigenetic level. In this paper, SOX2-OT was highly expressed in the patients with carotid atherosclerosis, suggesting that carotid atherosclerosis might lead to the alternation of SOX2-OT expression. In addition, the elevated SOX2-OT was correlated with the increased CIMT levels, which suggested that the progression of carotid atherosclerosis might contribute to the abnormality of SOX2-OT expression. Accumulated evidence reports the crucial roles of SOX2-OT in the regulation of tumors and various diseases. It is reported that the expression of SOX2-OT is enhanced in lung cancer, gastric cancer, and esophageal cancer –24, whicIn the present report, the clinical significance of SOX2-OT was further revealed. The finding of ROC curve implied that SOX2-OT might be a biomarker to differentiate the patients with carotid atherosclerosis from healthy cohorts with desirable specificity and sensitivity. In addition, the expression of SOX2-OT in the patients with good outcomes was reduced, which revealed that the expression of SOX2-OT might change with the development of carotid atherosclerosis. Besides, the K–M analysis discovered that patients with high SOX2-OT levels had a high possibility of suffering cardiovascular events, indicating that overexpression of SOX2-OT is a risk of poor outcomes. In addition, the result of Cox regression analysis indicated that SOX2-OT might be an independent prognostic biomarker in predicting the outcome of patients with carotid atherosclerosis. As reported by previous publications, several lncRNAs have the probability of being markers in chronic vascular diseases. In coronary atherosclerosis, Qual et al. discern that PVT1 is an independent factor influencing disease development . The ROCIn total, an enhancement of SOX2-OT expression in patients with asymptomatic carotid atherosclerosis was evaluated. The upregulation of SOX2-OT was associated with the ascended CIMT levels. Moreover, SOX2-OT might be a predictive indicator of the identification and progression of carotid atherosclerosis. This study discovered a novel biomarker in carotid atherosclerosis and this finding might be beneficial to the screen and predict patients with asymptomatic carotid atherosclerosis.


We previously showed that feeding selenium-enriched alfalfa hay to weaned beef calves diversified the nasal microbiome and improved health and growth in the feedlot, resulting in greater carcass weight and quality at slaughter. The objective of the current study was to see whether selenium supplementation of dams during various pregnancy trimesters similarly improves health and performance. Maternal supranutritional selenium supplementation during pregnancy increases selenium concentrations in calves, but is more effective the shorter the time period between maternal supplementation and assessment. Thus, calves should be fed selenium-enriched forages again at weaning in order to see a benefit in the nasal microbiome abundance and diversity, and consequently, in carcass measurements at slaughter.p < 0.01). At weaning, only calves from TR-3 mothers had higher WB-Se concentrations compared with calves from CTR mothers (p = 0.02), and no significant differences in nasal microbiome abundance and diversity or nasal microbiota were observed. In the feedlot period, morbidity was low, and no differences were observed. At slaughter, no differences in carcass weight and quality were observed. In conclusion, Se supplementation of pregnant cows is effective for increasing WB-Se concentration of newborn calves, and the increase can be sustained until weaning for calves born to TR-3 dams. However, the increase in WB-Se concentrations is small and does not result in beneficial changes in the nasal microbiome. Thus, calves should be fed Se-biofortified forages again at weaning in a preconditioning program in order to diversify the nasal microbiome prior to entering the feedlot.We previously reported that feeding Se-biofortified alfalfa hay to weaned beef calves in a preconditioning program increases whole-blood Se (WB-Se) concentrations and nasal microbiome abundance and diversity during the preconditioning period, decreases morbidity and mortality during the feedlot period, and increases carcass weight and quality at slaughter. The objective of the current study was to see whether similar improvements can be achieved through Se supplementation of dams during various pregnancy trimesters. In a two-year experimental study, 80 Angus-cross cows received once-weekly Se-yeast boluses containing 105 mg of Se, during either the first (TR-1), second (TR-2), or third (TR-3) pregnancy trimester, or were not bolused (CTR). Whole-blood Se concentrations were higher from CTR, to TR-1, to TR-2, and to TR-3 in newborn calves (all  Selenium (Se) in an essential trace mineral for cattle that plays an important role in growth and health. Therefore, Se-deficient soils represent unique challenges for cattle producers in terms of cattle management. Clinical Se deficiency, indicated by a whole-blood (WB) Se < 50 ng/mL, can lead to nutritional myodegeneration (white muscle disease) and death, especially during periods of high growth rates such as in young animals ,2. SubclThe Northwest region is among those regions with the lowest amounts of Se in soils and plants . In mostA major cause of morbidity and mortality in U.S. feedlot cattle is bovine respiratory disease (BRD) complex ,18. We pThe objective of the current study was to evaluate whether similar changes in calves can be achieved by administering Se boluses to their dams during different trimesters of pregnancy . We chose Se boluses rather than Se-biofortified alfalfa hay, as one can verify the amount of Se from selenized yeast ingested using boluses; however, this method is more labor-intensive than feeding Se-biofortified forages. Similar to our previous studies, we used Angus and Angus-cross cows that also had access to sodium selenate in a salt–mineral mix, at concentrations recommended by the US-FDA for preventing clinical Se deficiency. We hypothesized from our prior studies that Se requirements may differ among the three trimesters of pregnancy. Furthermore, we hypothesized that maternal Se supplementation may impact fetal programming . Selenium-responsive fetal programming may also impact outcomes long after the supplementation period has ended—e.g., changing calf growth and health prior to weaning as well as in the feedlot—and may affect carcass characteristics at slaughter. To our knowledge, this is the first study that examines the impact of maternal Se supplementation during different trimesters of pregnancy rather than focusing on the last weeks of pregnancy or throughout pregnancy.The Oregon State University Animal Care and Use Committee reviewed and approved the experimental protocol (ACUP Number: 2019–0056). The study design was a prospective clinical trial of a 2-year duration (May 2019–June 2021), and included calves born to beef cows that were bolused once weekly with Se yeast during various trimesters of pregnancy . Cows weForage Se concentrations in this region range from 0.06 to 0.11 mg/kg (DM basis) ,20. All ®” (CAB). Besides consistent sizing, the CAB certification, indicating superior carcass quality, requires a maximum of 1-inch fat thickness, superior muscling, a lack of quality deficits, and a minimum of 5.8% evenly distributed intramuscular fat content of the LD muscle with medium-to-fine marbling texture.After calving, all cows and calves were maintained on pasture and hay at the Soap Creek Ranch in Corvallis, OR, and had free-choice access to a mineral supplement containing 120 mg/kg Se from sodium selenite. Routine farm management practices, including vaccinations and deworming, were the same for all treatment groups. Health status was monitored during the pre-weaning period . The calves were weaned in October 2020 between 190 and 245 days of age, with a mean ± STDev of 224 ± 14 days. The best steers were kept for a beef production course (“Steer-a-Year”) and the best females were kept as replacement heifers. The remaining calves were shipped six weeks later (November 2020) to a commercial feedlot  in Burbank, WA. The feedlot rejected the smallest calves, which had to be sold privately. Calves were slaughtered in June 2021 at a commercial facility  between 439 and 495 days of age, with a mean ± STDev of 474 ± 14 days, and carcass weight and characteristics were determined. Carcasses were weighed and graded. Eight carcasses were classified as “Certified Angus BeefWhole-blood samples were collected within 12 to 48 h of birth (day 0), at 14 days of age, at 60 days of age, and again at weaning, into evacuated 2-mL EDTA tubes  and stored on ice until they were frozen at −20 °C. Frozen whole-blood samples were shipped on dry ice to Utah Veterinary Diagnostic Laboratory, a commercial laboratory in Logan, UT, to measure concentrations of WB-Se. In short, 750 µL of whole blood was mixed with 750 µL of trace-metal-grade nitric acid in a 10-mL digestion tube. Next, the blood was digested for 2 h at 90 °C in the cap-sealed tube. After cooling down, the contents were transferred to another trace-metal-free tube. Next, 1 mL of the digested blood was transferred into another trace-metal-free tube, which already contained 9.0 mL of ultrapure water, to form a 5% nitric acid matrix. After vortexing, the sample was analyzed on an inductively coupled argon plasma emission spectrometer  using a 4-point standard curve. A pooled QC sample dissolved in a 5% nitric acid matrix was analyzed after every 5 samples. If the pooled QC sample fell within ±5% of the reference value, the WB-Se analysis was considered acceptable.n = 10 , TR-1 n = 9 , TR-2 n = 11 , TR-3 n = 10 . Nasal swabs  were collected to assess the nasal microbiome at weaning (6.5 to 8 months of age). The nasal swabs, which were sterile and wrapped individually, were inserted approximately 10 cm into the ventral meatus of the nares, twirled to collect a mucosal swab, and then placed into individual sterile containers . Care was taken to avoid any contamination or human contact with the plastic stick. All swabs were then frozen at −80 °C within 4 h of collection in their collection tubes. Sterile swabs (negative controls) were processed in the same manner.Based on the uniform genetic background of cows ), ten calves were chosen from each group for assessment of nasal microbiota. The resulting calf group demographics from these cows were as follows: CTR group A MoBio Power soil DNA isolation kit  was used per the manufacturer’s instructions, to extract microbial DNA from the nasal swabs. The V4 region of the 16S rRNA gene was amplified with primers 515F (50-GTGCCAGCMGCCGCGGTAA-30) and 806R (50-GGACTACVSGGGTATCTAAT-30) at the MR DNA Laboratory  and sequenced on an Illumina MiSeq instrument at the MR DNA Laboratory .https://qiime2.org/, accessed on 25 May 2022) platform was used to process the sequencing data [The QIIME 2 v 2018.6  with a cut-off of α = 0.05 and an LDA score >2.0 [p ≤ 0.05 and a tendency at 0.05 < p ≤ 0.10.QIIME2 was used to generate principal coordinate analysis (PCoA) plots for weighted and unweighted UniFrac distances. In order to find significant differences in microbial communities between groups, an ANOSIM  test was performed on the weighted and unweighted UniFrac distances. We compared bacterial taxa between groups using the non-parametric Mann–Whitney U test. The ore >2.0 . The stap-values for time, treatment groups, and their interaction were all <0.0001. Selenium supplementation of dams  using weekly Se boluses during different trimesters was effective in increasing WB-Se concentration at birth in calves from all three Se-supplementation groups  compared with the calves of control dams . The effectiveness in increasing the WB-Se concentrations of calves was greatest when dams received Se supplementation in the last trimester (418 ± 8 ng/mL); medium increases were observed when dams received Se supplementation in the second trimester (250 ± 9 ng/mL); and the smallest increases were observed when dams received Se supplementation in the first trimester (169 ± 8 ng/mL).The concentrations of WB-Se at birth (0 days), 14 and 60 days of age, and at weaning are shown in p vs. control = 0.02). Smaller increases for a shorter time span were observed in the calves of TR-2 dams, as higher WB-Se concentrations were observed until 60 days of age . The smallest increases for the shortest time span were observed in the calves of TR-1 dams, as higher WB-Se concentrations were observed until 14 days of age .The length of effectiveness in increasing the WB-Se concentrations of calves was greatest when dams received WB-Se concentrations in the last trimester. Higher WB-Se concentrations were observed until weaning in the calves of TR-3 dams . Sex differences were also observed at 14 days of age , and at 60 days of age . In contrast, sex differences were not significant at weaning .Female calves had higher WB-Se concentrations than male calves at birth  and then increased back to 132 ± 7 ng/mL at weaning. Many calves, especially male calves, without recent maternal Se supplementation had WB-Se concentrations indicating subclinical Se deficiency .A total of 11 of the 15 male CTR-calves, 6 of the 14 male TR-1 calves, 4 of the 8 female CTR calves, 3 of the 7 female TR-1 calves and 1 each of the 11 and 10 male TR-2 and TR-3 calves, respectively, during the repeated sampling times, had WB-Se concentrations below 100 ng/mL . As the p = 0.21) and are shown in p = 0.04). The calves of TR-2 dams had lower weaning weights than the calves from CTR dams (p = 0.04), TR-1 dams (p = 0.06), and TR-3 dams (p = 0.02). Male calves at each sampling time had higher body or carcass weights than female calves  with the difference between weights increasing (sex-by-time interaction: p = 0.05). The calves of medium-frame-size cows had higher body weights than the calves of small- or medium-frame-size cows (p = 0.01), with no significant differences among sampling times (cow-frame-by-time interaction: p = 0.17).The body weights at birth, 14 days, 60 days, weaning, and at slaughter of calves of the four calf groups were similar (group main effect: p = 0.09) and a lower proportion with a CAB certificate  compared with the other two Se-supplemented groups (TR-2 and TR-3) combined. No other differences in carcass characteristics were observed .The results of the carcass characteristics of the calves from the four groups are reported in The nasal microbiome was collected at weaning. Sequencing of the bacterial 16S rRNA gene in the samples yielded 3,062,213 quality sequences . Rarefaction analysis was performed at a depth of 30,450 sequences. There was no significant difference in nasal microbiome diversity in calves at weaning based on the treatment of dams with Se-yeast boluses  during the first (TR-1), second (TR-2), and third (TR-3) trimester of gestation combined, compared with the control calves (data not shown). There was also no difference in nasal microbiome diversity among calves of the Se-treatment groups (TR-1 vs. TR-2 vs. TR-3) at weaning (data not shown). Subsequently, we reanalyzed the data from 11 calves from control cows and cows supplemented, during the first (TR-1) trimester of gestation, that had WB-Se in the range of 0.06 to 0.13 ng/mL, and compared the with 10 calves from TR-2 and TR-3 groups that had WB-Se in the range of 0.15 to 0.21 ng/mL. There was no significant difference in nasal microbiome alpha diversity in calves at weaning based on their WB-Se concentration in these two ranges . This waunweighted = 0.0258, p = 0.22; Rweighted = 0.0078, p = 0.38) of UniFrac distances were not significant.The nasal microbiome from the nasal swabs of calves at weaning also did not differ for beta diversity measures, as shown by principal coordinate analysis of UniFrac distances. Beta diversity looks at differences in the bacterial species between two groups of calves. Unweighted UniFrac distance measures differences in the number of ASV’s present, whereas the weighted UnifFrac distance accounts for differences in the number of bacterial species as well as the relative abundance of different bacterial species ,34. Prinp > 0.05)  .The objective of this study was to evaluate the effect of maternal supranutritional Se supplementation during various trimesters of pregnancy on the Se status, growth, nasal microbiome at weaning, and carcass characteristics of their calves. To our knowledge, this is the first study which examines the impact of maternal supranutritional Se supplementation during various trimesters of pregnancy on calf performance and health. In two previous studies, we supplemented dairy  and beefIn our previous studies, we did not follow WB-Se concentrations of calves beyond the first 48 h in beef calves  or 14 daThe current guidelines for WB-Se concentrations in calves (0 to 30 days of age) suggest that WB-Se concentrations of 100–250 ng/mL are adequate [Maternal, supranutritional Se-yeast supplementation of TR-3 cows did not impact growth performance. We previously reported higher birth weights of dairy calves whose dams were supplemented weekly with Se yeast for the last 8 weeks of pregnancy , but notp = 0.07). In addition, we previously reported that the genes involved in collagen formation were downregulated in the muscle of newborn beef calves of TR-2 vs. CTR cows [longissimus dorsi muscle, indicating a potential benefit for optimal muscle function and contraction [Given the lower weaning weights of calves from TR-2 cows, maternal Se supplementation during the second trimester also may not be beneficial for the muscle growth of calves. The hot carcass weight of the calves from TR-2 cows tended to be lower compared with the calves from CTR cows ; this number was lower in calves fed Se-biofortified alfalfa hay during the preconditioning period . In our Limitations of this study include the fact that that not all calves could be followed through the feedlot to assess performance, as some female calves were retained as seed stock, and male calves were also retained for a production class . The favIn conclusion, we cannot rely on Se supplementation of the cow during pregnancy to carry the calf through to the feedlot. Ultimately it is best to provide Se supplement to calves again at weaning, as we did in our previous studies, in order to see a benefit in nasal microbiome abundance and diversity. The timing of Se supplementation appears critical, as WB-Se concentrations and the physiological effects of Se attenuate the longer the time between the last Se application and assessment. Thus, calves should be fed Se-biofortified forages again at weaning in order to diversify the nasal microbiome prior to entering the feedlot.


Multiple Sclerosis (MS) is an inflammatory disorder wherein the myelin of nerve cells in the central nervous system is damaged. In the current study, we assessed the effect of Dapsone (DAP) on the improvement of behavioral dysfunction and preservation of myelin in the cuprizone (CPZ) induced demyelination model via targeting Nrf2 and IKB. MS was induced in C57BL/6 mice through diet supplementation of CPZ (0.2%) for 6 weeks, and DAP  was administered for the last 2 weeks of treatment. Pole test and rotarod performance test, LFB and H&E staining, and Immunohistochemistry (IHC) staining of p-Nrf2 and p-IKB were performed. Furthermore, superoxide dismutase (SOD) and nitrite were measured.P<0.001). Pole test showed that CPZ increased latency time to fall (P<0.0001) but the latency to reach the floor in the DAP-CPZ group was significantly shorter (P<0.0001). Rotarod performance test showed the effect of CPZ in reducing fall time in the CPZ group (P<0.0014); however, DAP significantly increased fall time (P=0.0012). In LFB staining, DAP reduced demyelination induced by CPZ. CPZ significantly decreased p-Nrf2 and elevated p-IKB levels compared with the control group (P<0.0001), but in DAP-treated groups markedly modified these changes (P<0.0001). CPZ increased the brain nitrite levels and reduced SOD activity, but in DAP-treated considerably reversed CPZ-induced changes. DAP treatment prevented body loss induced by CPZ (These data support the suggestion that the beneficial properties of DAP on the CPZ-induced demyelination are mediated by targeting Nrf2 and NF-kB pathways. Multiple sclerosis (MS) is a long-lasting autoimmune demyelinating disease of the brain and spinal cord that produces a variety of symptoms such as fatigue, incoordination, blurred vision, and psychiatric problems . Current+2 in mitochondrial matrix and cytosol and lipid peroxidation  (CPZ), a copper (Cu) chelator, has neurotoxic effects which can damage the white matter of the CNS . In 1966xidation , 13, andxidation . One of yte loss . So, CPZyte loss .2) is a transcriptional factor that controls the expression of anti-oxidant proteins and protects the cells against oxidative stress during inflammation and injury (2 present in the cytosol is inactivated by binding to kelch-like ECH-associated protein 1 (Keap1). During oxidative stress injury, Keap1 is oxidized and dissociated from the Keap1-Nrf2 complex, allowing Nrf2 to be activated by phosphorylation, becoming the biologically active form. Once activated, p-Nrf2 goes to the nucleus and increases the expression of antioxidant factors by binding to consensus antioxidant response elements (AREs) (2-regulated antioxidants are quinine oxidoreductase-1 (NQO-1) and heme oxygenase-1 (HO-1)  . Some of1 (HO-1) . Some re reduced , 20. ThuPneumocystis carinii pneumonia, malaria, acne, and many dermatologic disorders , and increasing the activity of SOD and it can also decrease pro-inflammatory cytokines as well as the level of p-NF-kB (Dapsone (DAP), a diaminodiphenyl sulfone, exerts antimicrobial and anti-protozoa effects . This suisorders , 24. It isorders -28. An i p-NF-kB . 2 were also determined in the brains of animals. Based on these previous data, the purpose of this research was to evaluate the therapeutic effect of DAP in reducing the CPZ-induced demyelination in C57BL/6 mice by rotarod and pole tests. Some oxidative stress factors such as SOD and nitrite and the levels of p-IKB and p-NrfAnimals°C, 12 hr/12 hr light/dark cycle with free admittance to food and water. All trials were accepted by the Ethics Committee of Tehran University of Medical Science (TUMS), Tehran, Iran.Twenty-one adult male C57BL/6 mice (20-25 g) were attained from the Pasteur Institute . Mice were kept at 22±1 Induction of cuprizone-demyelination and dapsone treatmentTo induce demyelination, mice were fed 0.2% (w/w) CPZ  in their chow for six weeks , 30. Ani1. Control group: fed with standard diet for six weeks.2. CPZ group: fed with a diet supplemented with 0.2% (w/w) CPZ for six weeks.3. DAP-treated CPZ group: fed with a diet supplemented with 0.2% (w/w) CPZ for six weeks and intraperitoneal injection of DAP   dissolved in normal saline -33 for tBehavioral testsThe behavioral tests – pole test and rotarod performance test– were done on the last day of research to assess the locomotor coordination of all animals (training of mice was performed). Both teThe rotarod performance test was carried out with a rotarod device . The rotating rod had a 3 cm diameter and a height of 15 cm above the instrument floor. The speed was set at 25 rpm and 3 consecutive trials every 10 min were done. The animal was put on the rotating cylinder and was allowed to run. The latency to fall was recorded. The cut-off time of the test was 300 sec.Brain preparations (H & E) and myelin staining and immunohistochemistry (IHC) assays. The rest of the brains were placed in 1 ml of cold PBS  and homogenized by a homogenizer  and then, centrifuged for 20 min . The supernatants were used for nitrite and SOD assays.At the end of the study, mice were anesthetized with ketamine/xylazine . The brains of mice were carefully dissected, and half of them were fixed in 10% paraformaldehyde for hematoxylin and eosinMyelin staining°C with LFB. Then, the slides were washed with 95% ethyl alcohol, differentiated in 0.05% LiCO3 solution for 30 sec and 70% ethyl alcohol for 30 sec, and rinsed in DW. Later, slides were dehydrated and fixed in graded ethyl alcohol and dimethyl benzene. The samples were checked by a light microscope (Olympus-ckx53), and the degree of demyelination was evaluated using the ImageJ software.Luxol fast blue staining (LFB) was used for staining axons on formalin-fixed, paraffin-embedded CNS tissue segments. The oligodendroglial myelin sheath on neurons, containing lipids, discolor from blue to green, and the neuronal cells discolor to purple. In this study, LFB was done to observe the region of demyelination in the corpus callosum based on the previous studies . After dHematoxylin and eosin staining°C). Then 10% acetic acid and 85% ethyl alcohol were used for tissue differentiation, and the tissues were washed with distilled water. After soaking the slides in lithium carbonate solution and washing with water, the staining was completed with eosin solution. The samples were checked by a light microscope, and vacuolization and chromatolysis of neurons were evaluated. The fixed prefrontal cortex was embedded in a paraffin block. 5 µm sections were cut by a microtome and incubated with Harris’ hematoxylin solution . After washing the slides with TBS plus 0.03% Triton X-100, blocking was performed with 1% BSA in TBS. Then, samples were blocked with a peroxidase blocking agent. Next, they were incubated at 4 °C overnight with rabbit monoclonal p-Nrf2 (phospho S40) (1:100 dilution) antibody  and rabbit polyclonal p-IKB alpha (phosphor Ser32/S36) (1:100 dilution) antibody . After washing with PBS and incubating with 3% H2O2 , slides were incubated for one hour with goat biotinylated polyvalent secondary antibody (1:100 dilution) . The samples were stained with diaminobenzidine (DAB) (10 min) and water after washing with PBS. Counterstaining with hematoxylin  and dehydrating with alcohol and xylene was performed. Finally, after applying the coverslip, the slides were observed and assayed with a light microscope. To quantify the results, ImageJ software was used  using SPSS . The significance of the results was determined by one-way analysis of variance (ANOVA) followed by Tukey’s BodyweightP<0.05). Throughout the study up to 6 weeks, the bodyweight of the CPZ group remained significantly below that of the control group (P<0.001). DAP administration significantly reversed CPZ-induced weight loss in the last two weeks of the study compared with the CPZ group (P<0.001).The bodyweight of animals was evaluated every week . After oBehavioral testsPole testP<0.0001) though the latency was significantly shorter in the CPZ group treated with DAP than in the CPZ group (P<0.0001).The results of the pole test performed on the last day of the study are presented in Rotarod performance test P=0.0014), indicating that the CPZ-treated mice had lower balance and coordination than mice receiving a normal diet. Additionally, treatment with DAP significantly increased the time to fall (P=0.0012), suggesting that DAP administration improved motor coordination. The results from the rotarod performance test after 6 weeks are shown in Myelin staining (Luxol fast blue staining)P<0.0001) evidenced by significant reduction of the intensity of blue color at the end of the study. DAP treatment showed a significant protective effect on myelin injury induced by CPZ and ameliorated the demyelination process observed in the CPZ group (P<0.0001). The intensity of the blue color is close to the color observed in control, indicating a good state of myelination. The integral optical densities of blue color in the three experimental groups are shown in In this experiment, as shown in Hematoxylin and eosin stainingTo evaluate the oligodendrocyte and neuron structures, H & E staining of the prefrontal cortex was carried out. As shown in Immunohistochemistry staining of p-Nrf2 and p-IKB2 and p-IKB was done to estimate the effect of CPZ and DAP treatment on these transcription factors. As shown in 2 compared with the control group =182.8, P<0.0001), evidencing the existence of oxidative damage induced by CPZ. DAP administration significantly reduced the CPZ-induced alterations in the levels of p-Nrf2 (P<0.0001).Immunohistochemical staining of p-NrfP<0.0001), which means that CPZ induces the activation of the NF-kB signaling pathway. DAP treatment significantly reduced the levels of p-IKB (P<0.0001).Another transcription factor that is influenced by CPZ is NF-kB, which increases the expression of pro-inflammatory cytokines . IKB forNitrite level and superoxide dismutase activityP=0.0097), and DAP administration significantly reversed the CPZ-induced changes in nitrite (P<0.0001). Oxidative stress was measured by evaluating the activity of the antioxidant enzyme SOD in brain tissue. As indicated in P=0.0002). Then, DAP treatment significantly reversed this reduced SOD activity (P<0.0001). These data indicate that CPZ exposure induced oxidative stress injury, whereas DAP ameliorated the CPZ toxicity. in vivo models for studying myelin disorders, for example, experimental autoimmune encephalomyelitis (EAE) , bleomycin-induced idiopathic pulmonary fibrosis, and cardiotoxicity induced by doxorubicin via NF-kB targeting, leading to modulation of diverse oxidative stress and inflammatory mediators such as SOD, nitrite, MDA, and TNF-α  , virus-iis (EAE) , and CPZis (EAE) , 43. Theis (EAE) . Despiteis (EAE) , 45. Dapis (EAE) . DAP shois (EAE) . In thisnd TNF-α , 47, 48.ithelial . It has ithelial . In addiithelial . Our data showed that the 6-week food supplemented with 0.2% (w / w) CPZ resulted in a deficit of locomotor coordination in the pole and rotarod performance tests. Many observations using the same model illustrated that dysfunctions in behavioral and motor tests are related to the demyelination of neurons , 53. To In addition to the increase in nitrite production, CPZ reduced SOD activity in the brain. This reduction was reversed in the group treated with DAP for 2 weeks. It has been shown that activated microglia cause neurotoxic astrocytes and subsequently cause the death of nerve cells and oligodendrocytes . In addi2, a transcription factor, has a cytoprotective role via increasing the expression of antioxidant genes, for instance, the detoxifying enzymes NQO-1 and HO-1  associated with the critical role of this signaling in demyelination induced by CPZ. It has been evidenced that the Nrf2-deficient mouse exhibited a higher susceptibility toward CPZ toxicity and demyelination. DAP administration up-regulated the levels of p-Nrf2 and, consequently, could ameliorate oxidative stress and neuroinflammation. Accordingly, DAP administration could protect oligodendrocytes against oxidative stress injury by triggering the Nrf2 pathway. It has been evidenced that astrocytes and oligodendrocytes overexpressing Nrf2 are capable of saving neuronal cells from oxidative stress  reduced apoptosis in the spinal cord injury model in rats via decreasing the levels of caspase-8, 9, and 3 as well as acting as an antagonist of glutamate receptors, MPO activity, and improved motor function and coordination (Nrfand HO-1 . Amira ahe brain . Besidesl damage . The pree stress . Rios andination , 75. To 2 and NF-kB signaling pathways. Findings suggest that DAP may be a promising agent for remyelination, although clinical trials are necessary to assess the effectiveness of DAP in patients with MS.The present study offers evidence for the neuroprotective effect of DAP against demyelination and locomotor dysfunction induced by CPZ. The protective effect of DAP may be correlated to its antioxidant and anti-inflammatory effects protecting oligodendrocytes from the oxidative stress induced by CPZ. The mechanism of action seems to be linked to the modulation of NrfSS designed the research; AD, SS, and EK collected data; TN, AM-F, and LD analyzed data; AS and ES wrote the manuscript and created all the figures. All authors were involved in writing and revising the final manuscript. The authors declare that they have no conflicts of interest.


Low sFlt-1 (soluble Fms-like tyrosine kinase-1) and ET-1 (endothelin-1) levels have been reported in preeclamptic women using proton pump inhibitors.Here, we examined whether the proton pump inhibitor omeprazole could acutely reduce sFlt-1 and ET-1 , or increase free PlGF  in 20 women with confirmed preeclampsia. Primary outcome was specified as the difference in sFlt-1, PlGF, or CT-proET-1 after 4 days of omeprazole versus 20 preeclamptic women not receiving omeprazole.+3 weeks. Baseline sFlt-1 levels were identical in both groups, and the same was true for PlGF or CT-proET-1. After 4 days, sFlt-1 levels remained similar in women not receiving omeprazole compared with women receiving omeprazole, while the levels of PlGF and CT-proET-1 also did not differ between groups. Women receiving omeprazole had a similar prolongation of pregnancy after inclusion compared with those in the nonomeprazole group (median 15 versus 14 days). Except for a higher neonatal intubation rate in the nonomeprazole group , there were no differences in maternal/perinatal complications. Finally, making use of the placenta perfusion model, we established that both omeprazole and its S-isomer, esomeprazole, when maternally applied, reached the fetal compartment , while only esomeprazole inhibited placental sFlt-1 release.Mean maternal age was 30 years, and median gestational age was 30Administration of omeprazole to women with confirmed preeclampsia does not alter their circulating levels of sFlt-1, PlGF, or ET-1, arguing against a role of this drug as a treatment for this syndrome. Preclinical studies suggest that proton pump inhibitors lower sFlt-1 (soluble Fms-like tyrosine kinase) and ET-1 (endothelin-1) in women with confirmed preeclampsia, thereby upregulating the non-sFlt-1-bound levels of placental growth factor.Yet, when treating women with confirmed preeclampsia with 40 mg omeprazole daily, no acute effect on either sFlt-1, ET-1, or placental growth factor was observed nor did this drug prolong pregnancy.Making use of the placenta perfusion model, omeprazole was observed to reach the fetal compartment.Omeprazole does not alter the circulating levels of sFlt-1, ET-1, and placental growth factor in pregnancy, arguing against a role of this drug as a treatment for preeclampsia.Given the lack of an acute effect of omeprazole in preeclamptic women, despite its earlier observed effect after longterm treatment and in preclinical studies, possibly lowering of sFlt-1 by proton pump inhibitors requires long-term treatment, started before the onset of preeclampsia.Thus, proton pump inhibitors should now be explored as preventive therapy rather than curative treatment.2 Approximately 5% of all pregnancies are affected by this syndrome, which is defined as the new onset of hypertension in the presence of maternal organ and/or uteroplacental dysfunction after 20 weeks’ gestation.2 Because there is no curative treatment for preeclampsia other than placental delivery, current management entails maternal administration of corticosteroids to accelerate fetal lung maturation, blood pressure control, and seizure prophylaxis while weighing the maternal risk of developing severe complications such as eclampsia, renal insufficiency, pulmonary edema and hemolysis, elevated liver enzymes, low platelets syndrome, against the danger of iatrogenic prematurity.1Preeclampsia is a hypertensive syndrome unique to human pregnancy, with significant impact on maternal and fetal wellbeing worldwide.4 Ideally, mitigating the release of sFlt-1 and ET-1 or enhancing PlGF production should resolve the angiogenic imbalance, thereby delaying disease progression in women with preeclampsia. As such, current studies focus on novel therapies that could target these (anti-)angiogenic factors.5 Recently, proton pump inhibitors (PPIs) were shown to dose-dependently reduce sFlt-1 release in placental explants or trophoblast cells.6 PPIs are commonly prescribed for the treatment of gastric acid reflux and are considered safe for use in pregnancy. Following these observations, our group demonstrated that in gestational age (GA)-matched women with suspected or confirmed preeclampsia, PPI use was associated with lower sFlt-1 and ET-1 levels compared with women not using these drugs.7 Since most women (80%) in the former study were using omeprazole,7 we hypothesized that administering this PPI to women with confirmed preeclampsia could actively decrease the levels of circulating sFlt-1.The wide clinical variety and complexity of preeclampsia has tempered our ability to precisely understand the pathogenic mechanisms underlying this syndrome. Yet, a well-recognized phenomenon is that the placenta releases excessive amounts of sFlt-1 (soluble Fms-like tyrosine kinase-1) into the maternal circulation, which in turn binds to and reduces free PlGF  and vascular endothelial growth factor. The ensuing highly antiangiogenic state subsequently triggers the release of the vasoconstrictor ET-1 (endothelin-1), further inducing the widespread maternal endothelial dysfunction typically associated with this syndrome.8) in women with confirmed preeclampsia. In addition, making use of the ex vivo placenta perfusion model, we investigated the transplacental transfer of omeprazole  and its S-isomer, esomeprazole, and established whether they could effectively reduce placental perfusate levels of sFlt-1.In the present study, we performed a randomized controlled trial to evaluate whether omeprazole treatment could alter the circulating and cord blood levels of sFlt-1, PlGF and CT-proET-1  between 2018 and 2021. All participants gave written informed consent to participate in the trial, which was approved by the Erasmus MC Research Ethics Committee (MEC-2018-071).+0 and 34+6 weeks+days on the day of randomization, a singleton pregnancy, were ≥18 years of age, and were able to give written informed consent. Women were not included in the trial if they were using any PPI at time of randomization, if they had a contraindication or hypersensitivity to PPI use, if they were using medication that could interact with PPI, if there was fetal death or distress at time of inclusion or if the clinicians expected delivery within the next 48 hours. The diagnostic criteria were based on the International Society of the Study of Hypertension in Pregnancy 2018 criteria, which defines preeclampsia as new-onset hypertension accompanied by proteinuria and/or maternal organ dysfunction and/or uteroplacental dysfunction at or after 20 weeks of gestation.9Women were eligible to participate in the study if they had a confirmed diagnosis of preeclampsia, a GA between 207 a sample size of 20 patients in both the intervention and control group was estimated. This would achieve 80% power to test the 67% decrease in sFlt-1 level reported in the study by Saleh et al,7 with a 2-sided alpha of 0.05.Based on the standardized effect size (1.0) and SD (0.93) calculated from the previous study conducted by Saleh et al,Participants were allocated to receive omeprazole 40 mg, once daily or no omeprazole. Randomization was performed using an online, web-based sequence generator. Since the circulating biomarkers could significantly alter with advancing gestation, randomization blocks were stratified according to GA .Women allocated in the omeprazole group received 30 capsules that were packaged for oral administration, with no special storage conditions. The omeprazole capsules were packaged and labeled by the pharmacy at the Erasmus Medical Center, which distributed the packs to the other participating center upon request. We advised that capsules should be taken once daily, preferably in the morning on an empty stomach, according to the pack’s instructions. Women in the omeprazole group that did not deliver 30 days after randomization would receive an additional omeprazole pack (containing 30 capsules). We recommended that treatment should be continued from enrollment until delivery. Compliance was monitored on a daily basis, since most women were admitted at the maternity units. After birth, all omeprazole packages were collected and counted by a research team member, to confirm compliance. In the non-omeprazole group, patients would not receive PPI, and those that developed gastric acid reflux during the trial, were advised to receive different drugs for this purpose. If despite these medications participants maintained gastric reflux requiring a PPI, they would be excluded.At study enrollment, baseline data was entered on a web-based program by 2 independent researchers (R.I. Neuman and M.D. Baars).Since the primary outcome of our study was the effect of omeprazole on maternal sFlt-1 production, we do not expect a placebo effect owing to the lack of blinding.In both the non-omeprazole group and omeprazole group, serum and EDTA plasma was collected at baseline , and day 1, 2, 4, 8 followed by twice weekly (during routine blood measurements) until delivery. The collected blood samples were stored after centrifugation, at −80 °C until analysis. Analysis of sFlt-1, PlGF, CT-proET-1 was performed at the end of the study. Serum sFlt-1 and PlGF were measured using an automated analyzer , while plasma CT-proET-1 levels were analyzed using a Thermo Fisher Kryptor Compact Plus .The primary outcome of the study was specified as the difference in sFlt-1, PlGF, and CT-proET-1 levels at day 4 after enrollment between the omeprazole group and the non-omeprazole group. Secondary outcomes comprised the difference in angiogenic markers 8 days after PPI administration, and the difference in longitudinal course or cord blood levels between the omeprazole and non-omeprazole group. Maternal and fetal/neonatal complications were assessed between groups. Maternal complications included eclampsia, pulmonary edema, placental abruption, subcapsular liver hematoma, cerebral hemorrhage/edema or infarction, visual disturbances, acute renal failure  or postpartum hemorrhage (blood loss ≥1 L after delivery). Fetal/neonatal complications comprised of neonatal intensive care unit admission; small-for-gestational age infant ; endotracheal intubation; sepsis; respiratory distress syndrome; bronchopulmonary dysplasia ; necrotizing enterocolitis and fetal/neonatal death. All outcomes were recorded on the web-based trial database by 2 independent researchers (R.I. Neuman and M.D. Baars).Placentas of women who underwent an elective cesarean section with uncomplicated singleton pregnancies were collected immediately after delivery at the Erasmus Medical Center, Rotterdam, the Netherlands. Clinical characteristics were obtained from the patients’ electronic files. All women who donated their placenta gave approval through written informed consent. The study was exempted from approval by the local institutional Medical Ethics Committee according to the Dutch Medical Research with Human Subjects Law (MEC-2016-418 and MEC-2017-418).10 Perfusion experiments were conducted in healthy, term placentas. Maternal and fetal perfusion media consisted of Krebs-Henseleit buffer at 37 °C, supplemented with heparin  and aerated with 95% O2 to 5% CO2. The fetal circulation  was established by cannulating the chorionic artery and corresponding vein of an intact cotyledon. Maternal circulation  was created by placing 4 blunt cannulas in the intervillous space. At t=0, at a concentration of 10×Cmax, esomeprazole (50 µmol/L) or omeprazole (30 µmol/L), or no drug as a control were added to the maternal circulation. Samples of the maternal and fetal circulations were taken after 6 minutes and every 30 minutes until the end of the experiment (180 minutes) to determine the fetal-to-maternal (F/M) ratio and sFlt-1 concentration and were immediately stored at −80 °C. To establish good overlap between maternal and fetal circulations, antipyrine (100 mg/L) was added to the maternal buffer, while FITC-dextran  was added to the fetal circulation as a marker of integrity of the capillary bed. In order for an experiment to be successful, the F/M ratio of antipyrine had to be >0.75 and the maternal-to-fetal ratio of FITC-dextran <0.03 at t=180.The perfusion model used in the current study has been described by Hitzerd et al.2 and modifier pumps, a refrigerated autosampler, a convergence manager with back pressure regulator, and a column oven. Chiral separation was performed on an ACQUITY UPC2 Trefoil AMY1 column , maintained at 20 °C. The mobile phase was composed of 80% CO2 and 20% ethanol/methanol  and was used at a flow rate of 1.5 mL/minute. A make-up flow consisted of the same composition and was set at 0.2 mL/minute. The back pressure regulator was set at 120 bar with a temperature of 70 °C. Total analysis time was 7 minutes. The auto sampler temperature was set at 5 °C. Perfusate samples were thawed and centrifuged, after which 50 µL was transferred to a microcentrifuge tube (1.5 mL). Protein precipitation was carried out by adding 1 mL of IS solved in methanol (ca. 1 mg/L). Next, the samples were vortex-mixed and centrifuged at 14 000 rpm for 5 minutes. The supernatant was transferred to vials, and a 5 µL aliquot was injected into the column, using a partial loop of 10 μL in needle overfill mode. Detection was performed on a Waters Xevo TQ-s micro mass spectrometry equipped with an electrospray ionization source, operated in positive ionization mode. The applied mass spectometry parameters were as follows: capillary voltage 3.5 kV, cone gas flow 1 L/hour, source block temperature 150 °C, and desolvation temperature 350 °C at a desolvation gas flow of 650 L/hour. Data acquisition was carried out using selected reaction monitoring. Ion transitions were m/z 346.18 to 198.13 for R/S-omeprazole and m/z 349.17 to 198.14 for the internal standard (S)-omeprazole-D3. A cone voltage of 20V and collision energy of 10V were used for both transitions. Raw data sets were processed by Masslynx software 4.1 and TargetLynx 4.1 (Waters Corp).The concentrations of S-omeprazole and R-omeprazole were measured in the perfusate by using a Waters Acquity UPC2-mass spectometry/mass spectometry system , equipped with a binary solvent pump including the COt test or Mann-Whitney U test  was performed. For the comparison of categorical variables between 2 groups, Fisher exact or χ2 test was applied. We used linear mixed models to evaluate the longitudinal course of the angiogenic markers according to treatment group. To account for within-subject correlations, a random intercept for study subject was used. Time (days at which blood was taken) and GA at the first measurement were added as fixed effects. Baseline values (at day 0) were added as covariate. For the placenta studies, differences in sFlt-1 levels were studied using general linear model repeated measurements. A P<0.05 was considered statistically significant. Data were analyzed using IBM SPSS statistics  and R studio Statistical Software.Data are reported as mean (±SD or SEM) or median (interquartile range) for continuous variables and as number (percentage) for categorical variables. The normality of continuous variables was assessed using the Shapiro-Wilk W test. For the comparison of continuous variables between 2 groups, an unpaired Figure S1). Baseline characteristics of all participants are displayed in Table 2. Median GA at enrollment was 30 (28–31) weeks, which did not differ between the 2 groups (P=0.45)Between December 2018 and June 2021, a total of 87 women with preeclampsia were found eligible for inclusion, of whom 57 agreed to participate and were randomized. Of these, 50 were included in the outcome analysis of whom 26 were allocated to the non-omeprazole group and 24 to the omeprazole group . At baseline, levels of sFlt-1, PlGF, and sFlt-1/PlGF ratio were not significantly different between the omeprazole group (n=20) and the non-omeprazole group  nor did changes occur in the levels of PlGF  or the sFlt-1/PlGF ratio .Of the 50 women included for the outcome analysis, 10 were excluded from the primary outcome analysis either because they delivered within 4 days (n=9) or because biomarker measurements on day 4 were not performed according to protocol (n=1) (P=0.01) and a lower sFlt-1/PlGF ratio  compared with the non-omeprazole group (n=15). However, baseline sFlt-1 levels in these 31 women were also significantly lower (P=0.01) in the omeprazole compared with the non-omeprazole group, and the same was true for the sFlt-1/PlGF ratio (P=0.02) (data not shown). CT-proET-1 measurements were only performed in 31 women, of which the values did not differ between groups at baseline, or at 4 or 8 days after enrollment  displayed lower sFlt-1 levels , as displayed in Table P=0.70). Adverse maternal outcomes did not occur more often in the non-omeprazole group in comparison to women receiving omeprazole (Table P=0.02).Pregnancy outcomes were evaluated in all 50 included women. Mean GA at delivery was 33 weeks in the omeprazole group n=24), compared with 32 weeks in the non-omeprazole group .Of the 18 placentas of the women initially included in the perfusion study, 16 met the quality control criteria (success rate of 88%). Maternal characteristics and characteristics of the placentas and offspring are shown in P=0.01), as shown in Figure P=0.95).The maternal sFlt-1 release in esomeprazole-perfused placentas (n=5) was statistically significantly diminished in comparison to control placentas  and deleterious  effects in preeclampsia. In fact, a population-based register cohort involving 157.720 nulliparous pregnant women, recently reported an increased risk of overall preeclampsia and preeclampsia at term of PPI use during any point of pregnancy, while PPI use recorded after 28 gestational weeks was associated with a reduced risk of preterm and early preeclampsia.14 Based on this, a unifying concept might be that to exert beneficial effects in preeclampsia, PPIs should be used over a defined period, in close proximity to disease onset, and most likely for >2 weeks. Interestingly, when we perfused healthy placentas with esomeprazole, maternal effluent levels of sFlt-1 were significantly lower in relation to controls. This was not observed for omeprazole. Similarly, Onda et al6 noted that esomeprazole and lansoprazole induced the highest dose-dependent sFlt-1 decrease in primary trophoblast and endothelial cells, whereas omeprazole exerted more modest reductions in sFlt-1. The simplest explanation of these findings is that, for a given concentration, esomeprazole contains double the amount of the active S-isomer. Furthermore, in the present study, the transplacental transfer of S-omeprazole alone  tended to be higher than its transfer in the presence of R-omeprazole , providing yet another reason for a higher tissue exposure to the active isomer, and thus a stronger effect, following esomeprazole exposure. Simultaneously, the in vivo half-life of the S-isomer is longer,15 but this is unlikely to be of relevance during a 3-hour placental perfusion period. Nevertheless, such acute effects of the S-isomer, if occurring at all, seem to be of limited relevance, since both esomeprazole11 and omeprazole (present study) were unable to diminish sFlt-1 release in an in vivo situation. Here, an important question remains why PPI-induced sFlt-1 reductions have been observed in preclinical studies, while we and others were not able to confirm these findings in clinical trials. Remarkably, this pattern has also been witnessed with other drugs such as pravastatin or sulfasalazine, which have shown significant sFlt-1-lowering properties in both placental tissues and animal models, although these actions were not apparent when examined in human patients.20 One possibility is that the doses applied in experimental models were much higher compared to the concentrations administered in clinical studies. Indeed, when the Cmax equivalent of 40 mg esomeprazole (5 µmol/L) was applied in primary tissues, it did not induce a significant reduction in sFlt-1, in contrast with concentrations of 50 or 100 µmol/L.6 Increasing the dose  or applying these drugs intravenously might solve this problem, although there are limited data regarding pharmacokinetics and teratogenic risks for these concentrations. Indeed, since we observed a placental transfer of at least ≈40% to 50% to the fetal side, fetal drug levels may substantially increase when maternal blood concentrations are higher. In addition, it could be the case that the sFlt-1-diminishing effect of PPIs is simply insufficient to counteract the massive placental release when preeclampsia is already established. Therefore, we propose to explore PPIs as preventive therapy rather than curative treatment, for example, in combination with aspirin. In fact, a randomized-placebo controlled trial has already been initiated where the consequences of daily esomeprazole administration to women at high risk of preeclampsia will be assessed.5 We reported a substantially higher neonatal intubation rate in the nonomeprazole group compared with the omeprazole group. The nonsignificant higher baseline sFlt-1/PlGF ratio in the nonomeprazole group might partially explain higher disease severity and therefore more iatrogenic preterm birth in the latter group. However, the proportion of neonates admitted to the neonatal intensive care unit or presenting with other complications remained similar between groups, arguing against this hypothesis. Moreover, in our former analysis evaluating PPI users, a nonsignificant trend toward higher intubation rate was similarly observed in the non-PPI group compared with the PPI group .7 On the other hand, in the trial by Cluver et al,11 where a much larger population was evaluated, a difference in intubation rate between esomeprazole and placebo group was not found. Hence, caution is granted when interpreting these findings, since our study is underpowered for these outcomes.In this randomized trial of women with confirmed preeclampsia, daily administration of 40 mg omeprazole did not significantly reduce the circulating levels of sFlt-1 and CT-proET-1, or increase the levels of PlGF after 4 or 8 days of treatment. Additionally, we observed no alterations in the longitudinal course of these angiogenic markers in maternal blood or in their cord blood levels, when comparing women receiving omeprazole compared with those not using this drug. When evaluating pregnancy outcomes, maternal complication rate was not different in the omeprazole group versus the non-omeprazole group, and the same was true for neonatal complications, except for a higher proportion of neonates requiring intubation in the non-omeprazole group. The present study is the first to evaluate the immediate consequences of omeprazole administration on circulating angiogenic marker levels in women with preeclampsia. Our results argue against a direct effect of omeprazole on sFlt-1 release as an explanation of our previous observation that sFlt-1 levels were 67% lower in 40 women with suspected or confirmed preeclampsia using PPIs compared with 80 gestational-age matched women not on this treatment.Our study has several strengths. Importantly, our population accurately reflects the clinical characteristics of women with preterm preeclampsia, of which >70% had a sFlt-1/PlGF ratio ≥85. In addition, we were able to closely monitor all participants, leading to a high compliance rate in our trial. Since most women already delivered within 1 to 2 weeks after enrollment, a limitation of our study was the low number of women assessed for the serial course of the biomarkers between the omeprazole and non-omeprazole group. Nevertheless, this was a secondary outcome, similar to the maternal/neonatal adverse outcomes, whereas our power calculation was solely based on detecting a difference in sFlt-1. In summary, our findings argue against a role for PPIs as potential treatment for preeclampsia, since daily administration of this drug was unable to improve the maternal circulating profile of sFlt-1, PlGF, and ET-1 in women with established preeclampsia.We thank Roche Diagnostics for providing the kits to measure sFlt-1 (soluble Fms-like tyrosine kinase-1) and PlGF , and Thermo Fisher Scientific for providing the kit to measure CT-proET-1 .R.I. Neuman and A.H. van den Meiracker are supported by the Dutch Foundation Lijf en Leven.None.


Time restricted eating (TRE), limiting eating to a specific daily window, is a novel dietary intervention, but the mechanisms by which TRE results in weight loss remain unclear. The goal of the current study was to examine changes in eating patterns, sleep, and late-night eating, and associations with health outcomes in a secondary analysis of a 12-week self-selected TRE intervention.n = 11) were instructed to eat during a self-selected 8-h window, while the non-TRE group (n = 9) followed their typical eating habits. All participants logged oral intake using the myCircadian Clock mobile application throughout the entire intervention. Anthropometrics, HbA1c, an oral glucose tolerance test, and 2 weeks of actigraphy monitoring were completed at pre-intervention and end-intervention. Independent samples t-tests compared differences between groups. Data are presented as mean ± standard deviation.Twenty participants 18–65 years with BMI ≥25 kg/m2 completed the 12-week trial. Participants randomized to TRE (p = 0.006) and higher HbA1c . The TRE group significantly delayed the timing of the first eating occasion by 2.72 ± 1.48 h relative to wake time (p < 0.001) and advanced the timing of the last eating occasion by 1.25 ± 0.8 h relative to bedtime (p < 0.001). The non-TRE group, on average, maintained their eating pattern. Sleep measures did not change from pre- to end-intervention, however greater restriction of the eating window was associated with longer sleep duration at end-intervention . The TRE group significantly reduced the prevalence of late night eating (eating within 2 h of bedtime) by 14 ± 6% (p = 0.028) with 63% of participants completely eliminating late night eating at end-intervention.At preintervention, late night eating was significantly associated with higher fasting glucose (r = 0.59, A self-selected TRE intervention significantly shifted meal timing, reduced late-night eating while prolonging sleep duration.ClinicalTrials.gov, identifier: 03129581. Time-restricted eating (TRE), limiting eating to a specific daily window, is emerging as a novel weight management intervention. Several studies have shown that restricting adults with long habitual eating windows (15 or more hours per day) to a food intake window of 8–10 hours per day results in weight loss . In a ran = 116) assigned late TRE (12-8 pm eating window) and reported no significant difference in weight compared to eating over a 12-h window  vs. restricted eating (TRE: self-selected eating window of 8 h) intervention. We characterized the change in timing of eating and eating patterns from pre- to end-intervention, evaluated whether the TRE intervention reduced late-night eating, and examined associations between timing of eating and health outcomes. We hypothesized that the TRE intervention would reduce late-night eating and that earlier timing of the last eating occasion would be associated with improved metabolic health outcomes.This is a secondary analysis of the See Food Study, a randomized controlled trial examining the impact of TRE on body composition and glycemic outcomes NCT# 03129581). Details of the study have been previously reported in the primary outcomes manuscript (9581. Detth day). Participants were excluded if they were pregnant or anticipated becoming pregnant, nursing, or had any significant medical problems such as diabetes or cardiovascular disease based on a screening visit and laboratory evaluation.Participants were aged 18–65 years with BMI ≥ 25 kg/m  using the mobile application, the myCircadian Clock (mCC: www.mycircadianclock.org) throughout the entire study. Height, weight, body composition, oral glucose tolerance test, and hemoglobin A1c (HbA1c) were obtained at pre-intervention and 12-week end-intervention, and participants wore an ActiGraph Link  to monitor sleep and activity for 2 weeks just prior to randomization (pre-intervention) and for 2 weeks just prior to study conclusion .After informed consent, participants were randomized to either TRE or non-TRE intervention, stratified by sex and age (±45 years). The TRE group self-selected a daily 8 h eating window, such as 10 am-6 pm or 11 am-7pm, which they were instructed to consistently maintain for the 12 week intervention. During the 8 h eating window, the TRE group could eat Each participant was instructed to document all oral intake including food, beverages along with an identifying text entry, using the mCC mobile application from the pre-intervention period through Week 12 of the study. The data were time-stamped and adherence to logging (TRE and non-TRE group) and compliance to eating windows (TRE group) were derived from the mCC data. Criteria for adherence have been previously described , intermediate-starters (first eating occasion 3.0–5.5 h after waking) and late-starters (first eating occasion ≥ 5.5 h after waking). The timing of the last eating occasion was used to classified early-enders (last eating occasion ≥ 5.5 h before sleep), intermediate-enders (last eating occasion occurring between 3.0–5.5 h before sleep), and late-enders (last eating occasion occurring ≤ 3.0 h before sleep). The categorial classifications of the beginning and end eating windows were combined to create a composite meal timing classification. All potential meal timing classifications are described in the These classifications were also used to approximate different TRE patterns: early-TRE, late-TRE, or intermediate-TRE pattern. Early-TRE eating patterns had eating windows which were concentrated in the early portion of the day (early-starters and early-enders) and was defined as eating primarily in the early portion of the day (first eating occasion ≤ 3.0 h after waking and last eating occasion ≥ 5.5 h before sleep). Late-TRE was defined as eating toward the later portion of the day (first eating occasion occurring ≥ 5.5 h after waking and last eating occasion occurring ≤ 3.0 h before sleep). Intermediate-TRE was defined as delaying the first eating occasion and advancing the last meal of the day (first eating occasion occurring between 3.0–5.5 h after waking and last eating occasion occurring between 3.0–5.5 h before sleep). Eating pattern  was classified for participants at pre-intervention and end-intervention depending on the eating pattern that was followed for the majority of the time . Finally, we determined the frequency of late-night eating, as defined by eating ≤ 2 h before bedtime, at pre-intervention and end-intervention.The Actigraph Link , a triaxial accelerometer, was worn on the non-dominant wrist for 2 weeks at pre-intervention and again at end-intervention. Data were collected in 60 second epochs and scored using the Cole-Kripke algorithm. Participants reported their typical weekday and weekend bed and wake times, which were used to facilitate actigraphy scoring. Sleep variables calculated included bedtime, waketime, and total sleep time. The ActiGraph Link has been validated against a previously validated accelerometer for sleep and shown to have a high level of agreement for sleep/wake  .At pre-intervention and end-intervention, height and weight were measured by study staff. DXA  assessed body composition using standard positioning and imaging protocols.Markers of insulin sensitivity were measured at pre-intervention and end-intervention using an oral glucose tolerance test (OGTT) following an 8 h fast. After baseline sampling, 75 g glucose  was consumed orally and plasma glucose and serum insulin were sampled every 30 min over 2 h. Homeostatic model assessment of insulin resistance (HOMA-IR) was calculated as [fasting insulin (μU/ml) x fasting glucose (mmol/l)] / 22.5)  from OGTn = 9, TRE: n = 11). One individual was excluded from the non-TRE at end-intervention because they only provided 1 day of sleep data. In the TRE group, one individual did not have any meals logged during the sleep measurement period. Thus, the end-intervention analysis included 8 individuals in the non-TRE group and 10 individuals in the TRE group.An a priori power analysis found that 20 participants (11 in the TRE group and 9 in the non-TRE group) gave 80% power to detect differences in the primary study outcomes [see primary outcomes paper . SPSS Statistics 26 and R statistical software version 4.0.2 were utilized for analyses. Statistical significance was set at p < 0.05.Demographic characteristics were summarized with descriptive statistics. Independent samples p > 0.05). No participants were taking any medication for dysglycemia or weight loss treatment during the study. On average, participants had 8 ± 3.0 (mean± SD) valid days  at pre-intervention and 9 ± 2.5 days at end-intervention. There were no significant between group differences.Eleven participants completed the TRE intervention , and nine completed the non-TRE intervention . No significant differences were found between groups regarding sex, age, or pre-intervention BMI, insulin sensitivity, or eating window  and higher HbA1c . There were no other significant associations between health outcomes and the timing of last eating occasions. There were also no significant associations at end-intervention after accounting for pre-intervention values.We evaluated the relationship between late night eating and health outcomes at pre-intervention . Eating Between the TRE and non-TRE groups, there were no differences in the timing of eating occasions during the pre-intervention period. At end-intervention, the TRE group significant delayed the time of the first eating occasion of the day, both when expressed by absolute clock time and as hours since wake . The TREn = 17, 85%) started eating early in the day and ended eating late in the day . At end intervention, most of the participants in the TRE group  did not follow a consistent eating pattern. One individual in the TRE group shifted to a consistent Early-TRE eating pattern such that they began eating early in the day and finished eating early in the day on 55% of days. There were 3 individuals who followed a consistent Intermediate-TRE eating pattern where the first eating occasion was moderately delayed, and the last eating occasion was moderately advanced, on 54 ± 7% of days. Finally, no participants in the TRE group elected to follow a Late-TRE pattern. While most individuals  in the non-TRE group maintained the Early-Late eating pattern at end intervention, there were 2 individuals who shifted to an inconsistent eating pattern in non-TRE. Finally, there were 2 individuals in the non-TRE group who adopted a Late-TRE eating pattern where they delayed both their first and last eating occasions.Eating pattern was categorized using the timing of the first and last eating occasion of the day . At pre-p = 0.014). At end-intervention, there was a significant reduction in late-night eating in the TRE group  and all but 4 participants in the TRE group completely eliminated eating within 2 h of bedtime. The non-TRE group did not significantly change late-night eating habits compared to pre-intervention, with 9 individuals performing late-night eating 43 ± 27% of the time (p = 0.068). Change in late-night eating was not significantly correlated with sleep duration (p > 0.05).We investigated change in late-night eating, or the timing of the last eating occasion relative to bedtime (expressed as hours before bed) from pre- to end-intervention. At pre-intervention, all participants had at least one instance of eating within 2 h of bedtime. The non-TRE group ate within 2 h of bedtime 57 ± 19% of the time, whereas the TRE group had significantly fewer instances of late-night eating at pre-intervention .Pre-intervention values and change in actigraphy-estimated sleep variables are reported by group in In this examination of participants following a randomized-controlled trial of a self-selected TRE intervention, we found that the TRE group significantly delayed the first eating occasion of the day and advanced the last eating occasion of the day, with most participants following an Early or Intermediate TRE eating pattern in the TRE group. In contrast, most participants in the non-TRE group maintained an Early-Late eating pattern such that their food consumption occurred across the entire day. Notably, only 4 participants in the TRE condition were consistent across days in their eating pattern, while most participants had a combination of both Early and Intermediate eating.Findings from the current study suggest that TRE intervention does not worsen sleep, as we found no change in objectively measured sleep duration or timing from pre- to end-intervention for either group. Moreover, greater restriction of the eating window was associated with longer sleep duration. However, study participants on average obtained less than the recommended 7–9 h of sleep per night, suggesting insufficient sleep. As insufficient sleep is independently associated with obesity, insulin resistance, type 2 diabetes, and metabolic syndrome –27, a foLate-night eating was significantly reduced in the TRE group, with participants completely ceasing food consumption within 2h of bedtime at end-intervention. This was in contrast to the non-TRE group which did not change their late-night eating habits. Prior studies have shown that late-night eating is associated with obesity, dyslipidemia, hyperglycemia, and metabolic syndrome , consistvia melatonin in conjunction with both self-selected and assigned TRE schedules may help to better elucidate how TRE interacts with circadian rhythms to effect weight loss.The field of TRE suggests restricting eating to a certain time of day affects daily activity-rest rhythms and the intrinsic circadian clock . SpecifiStrengths of the current study include objective measurement of sleep variables, real-time assessment of dietary timing, and a randomized, age- and sex-matched non-TRE control group. Having the control group provided critical comparison, as several participants still shifted their eating patterns despite being instructed to maintain their usual eating habits. Limiting factors include the small sample size, which limited our power and reduced our ability to control for potential covariates in analyses, though the current sample is similar to many of the existing TRE studies , 11. As In summary, our data show that during self-selected TRE  most participants followed an Early and/or Intermediate eating pattern and significantly reduced their late-night eating. The TRE intervention did not negatively impact sleep, and those with greater restriction of their eating window also had longer sleep duration. We recommend applying the meal timing classification methodology to future trials of TRE in larger sample sizes. Future research examining eating variability, directly intervening on sleep habits, and comparing Early vs. Late TRE, as well as Intermediate TRE as a novel framework in larger samples may help elucidate mechanisms responsible for the effect of TRE on weight loss in individuals with obesity.The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.The studies involving human participants were reviewed and approved by University of Minnesota IRB. The patients/participants provided their written informed consent to participate in this study.LC, SS, and JB: conceptualization. LC, DM, SP, and EM: methodology. SS and JB: formal analysis and writing–original draft preparation. LC: investigation and funding acquisition. SS, JB, EM, SP, DM, and LC: writing–review and editing. All authors have read and agreed to the published version of the manuscript.This work was supported by the Healthy Foods Healthy Lives program (17SFR-2YR50LC to LC) and the National Institutes of Health . The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.Author SP has authored the book The Circadian Code, for which he receives author royalties and in which he specifically recommends time restricted eating. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.